Herpes simplex virus (HSV) 1 and 2 are common ?-herpesviruses that establish lifelong latency. While most HSV infections of mucosal tissues are relatively benign, there are rare cases of encephalitis and HSV infections of the cornea can produce inflammatory pathology known as herpes stromal keratitis (HSK). HSK frequently involves recurring infections, often over years, produced by virus that reactivates in sensory ganglia that then travels to the cornea, producing scarring which can eventually lead to blindness. In the U.S. there are 60,000 cases of HSK/annually and HSV remains the leading infectious cause of blindness. Recurrent HSV infections in mucosa and the eye stem from reactivation of latent virus in neurons followed by anterograde transport in neuronal axons, a process by which virus particles hitchhike on kinesin motors that ferry virus from neuron cell bodies to axon tips. Our research will study two stages of anterograde transport. The first stage involves assembly of virus particles in the cytoplasm, followed by staging or sorting of these virus particles for transport into neuronal axons. The second stage involves transport of virus particles by kinesin motors along microtubules within axons. Our studies of first stage of this process will focus on two HSV membrane proteins gE/gI and US9 that cooperate to promote the assembly of virus particles and the polarized sorting of particles into axons. We recently demonstrated that HSV gE-/US9- double mutants were unable to assemble enveloped virus particles and, instead, viral capsids accumulated on cytoplasm membranes. There was also evidence that gE/gI and US9 participate in a subsequent process, involving sorting of enveloped virions into axons. These observations represent a novel example of neuron-specific defects in virus assembly and sorting in neurons and represent a new paradigm for how gE/gI and US9 function in neurons. The research in Aim 1 will test two hypotheses: i) gE/gI and US9 promote virus assembly by collecting other viral assembly proteins on cytoplasmic membranes that are sites of virus envelopment and ii) following envelopment, gE/gI and US9 trafficking sequences promote sorting of virus particles into axons. The molecular mechanisms involved in gE/gI- and US9-mediated assembly and sorting will be investigated using novel high resolution imaging techniques coupled with a panel of viral mutants and biochemical assays. In Aim 2, we will study the second stage of anterograde transport addressing two fundamentally important questions: i) which of the many kinesin motors transport HSV particles in axons and ii) which viral proteins tether onto kinesins? To address these questions, we will take advantage of recent advances in our ability to transduce neurons using baculoviruses to deliver fluorescent cargo molecules and kinesins, ?split kinesins? and miRNAs to silence kinesins allowing us to determine which kinesins are functionally important for HSV anterograde transport. Based on our recent identification of certain kinesins that transport HSV, we now have the opportunity to identify HSV proteins that tether onto these kinesin motors.